4,5,4′-Trihydroxychalcone, 8,8′-(ethene-1,2-diyl)-dinaphtalene-1,4,5-triol and rutin from Gynura segetum inhibit phagocytosis, lymphocyte proliferation, cytokine release and nitric oxide production from phagocytic cells

Background Gynura segetum is used traditionally to treat various ailments related to the immune system, which include cancer, inflammation, rheumatism, diabetes, hypertension, and viral infections but little studies have been carried out to validate their ethnopharmacological aspects. In this study the immunosuppressive effects of G. segetum and its constituents were investigated. Methods Isolation of compounds from G. segetum leaves was conducted using vacuum liquid chromatography (VLC) and column chromatography (CC). Two new compounds, namely 4,5,4'-trihydroxychalcone and 8,8'-(ethene-1,2-diyl)-dinaphtalene-1,4,5-triol, together with stigmasterol and β-sitosterol were isolated from G. segetum methanol extract and their structures were determined spectroscopically. The presence of gallic acid and rutin in the extract was determined quantitatively by a validated HPLC method. G. segetum methanol extract and its constituents were investigated for their effects on chemotaxis, phagocytosis, β2 integrin (CD18) expression, and reactive oxygen species (ROS) of polymorphonuclear leukocytes (PMNs), lymphocytes proliferation, cytokine release and nitric oxide (NO) production of phagocytes. Results All the samples significantly inhibited all the innate immune responses tested except CD 18 expression on surface of leukocytes. Among the samples, 8,8'-(ethene-1,2-diyl)-dinaphtalene-1,4,5-triol exhibited the strongest inhibitory on chemotaxis, phagocytosis, ROS and NO production. The compound exhibited exceptionally strong inhibitions on ROS and chemotaxis activities with IC50 values lower than the positive controls, aspirin and ibuprofen, respectively. 4,5,4'-Trihydroxychalcone revealed the strongest immunosuppressive activity on proliferation of lymphocytes (IC50 value of 1.52 μM) and on release of IL-1β (IC50 value of 6.69 μM). Meanwhile rutin was the most potent sample against release of TNF-α from monocytes (IC50, 16.96 μM). Conclusion The extract showed strong immunosuppressive effects on various components of the immune system and these activities were possibly contributed mainly by 4,5,4'-trihydroxychalcone, 8,8'-(ethene-1,2-diyl)-dinaphtalene-1,4,5-triol and rutin.

Keywords: Gynura Segetum, 4,5,4'-Trihydroxychalcone, 8,8'-(Ethene-1,2-diyl)-dinaphtalene-1,4,5-triol, Rutin, HPLC analysis, Immunosuppressive effects Background Modulation of the immune system is necessary in the treatment and management of those diseases due to defect or malfunction of the immune systems [1]. Immunosuppressive drugs inhibit the components of the immune system and they are used widely in the treatment of autoimmune diseases, inflammatory disorders and control pathological immune reaction of transplanted organs, while immunostimulant drugs activate the immune system and are widely used for the treatment of infectious diseases, cancer, allergy and immunodeficiency [2]. There are several chemical immunomodulators used in therapeutics but many of them have serious side effects. Aspirin and ibuprofen are among the non-steroidal antiinflammatory drugs (NSAIDs) which can cause gastric and intestinal mucosal problems [3]. Corticosteroids, which have long been used as immunosuppressants have showed various side effects such as fluid retention, weight gain, diabetes, increased skin fragility and reduced bone marrow. Cyclosporin A, the most widely used immunosuppressant in transplanted rejection treatment, can cause nephrotoxicity, tremors, hypertension and gingival hyperthrophy. Interleukin-2 causes many side effects, such as hypotension, tachycardia and edema. New and safer drugs are required as alternatives and phytochemicals will continue to be a replenishable source of new and safe immunomodulatory agents [4].
In an effort to look for safer immunomodulators, many secondary metabolites such as terpenoids, phenolics, and alkaloids have been investigated for their ability to modulate the immune system. The extracts of many herbs such as Panax ginseng, Tinospora cordifolia, Phyllanthus debilis, Centella asiatica Trigonella foenum graecum, Pouteria cambodiana, Picrorhiza scrophulariiflora and Garcinia mangostana were able to upregulate or downregulate both innate and adaptive arms of the immune response [5][6][7]. The chemical constituents of these plants are potential new sources of immunomodulating agents. The assessment of immunological activities of phytochemicals can be based on their effects specifically on the various component and functions of the immune system. There have been a surge in interest to prospect for natural immunomodulators from plants which have been used traditionally to treat many immunological disorders.
The genus Gynura is an annual herb belonging to the family Asteraceae. It consists about 20 species, which is distributed in Africa, Australia, and various parts of Asia especially in Southeast Asia, more particularly in Indonesia, Malaysia, and Thailand [8]. Among them, Gynura procumbens known as 'Sambung Nyawa' and G. segetum known as 'Daun Dewa' are used traditionally to treat various ailments which include inflammation, rheumatism, cancer, viral infections, diabetes and hypertension [9,10]. Many studies have been carried out to validate the pharmacological activities of G. procumbens. However, scientific studies on G. segetum were rarely reported. The leaf extract of G. segetum (Lour.) Merr. revealed potent anti-angiogenic activity which might lead to decrease tumor growth [11]. Phytochemical studies of the leaves of G. segetum showed the presence of flavonoids, tannins, saponins, terpenes, and alkaloids [9]. There is no effort yet to validate the traditional use of G. segetum leaves to treat diseases related to the immune system. Therefore, the present study was performed to determine the effects of the chemical constituents of the methanol extract of G. segetum leaves on phagocytic activities of polymorphonuclear leukocytes (PMNs), cytokine release, lymphocytes proliferation, and nitric oxide (NO) production of phagocytes.

Collection of plant material
Gynura segetum leaves was obtained from Yogyakarta, West Java, Indonesia in May 2012. A botanist of the Herbarium of Universiti Kebangsaan Malaysia (UKM) authenticated the plant material and a specimen (voucher number UKMB 29987) was deposited at the herbarium.
Cell culture RAW 264.7 murine macrophages cells were maintained in DMEM supplemented with 10% of FCS, and 100 units/mL each of of penicillin and streptomycin were grown at 37 0 C and 5% CO 2 in humidified air [12].
HPLC quantitative analysis of the methanol extract of Gynura segetum HPLC quantitative analysis of the methanol extract of the plant was carried out using the modified method of Seow et al. [9]. The methanol extract and the reference standards (rutin and gallic acid) were dissolved in methanol to obtain 10 and 1 mg/mL solutions, respectively. The diluted solutions were filtered through Millipore Millex PTFE membrane (0.45 μm) (Maidstone, Kent, UK). HPLC qualitative and quantitative analyses of the filtered solutions were carried out by using a C-18 column Xbridge (250 mm × 4.6 mm i.d., 5 μm) (Waters, Ireland) with the following method: mobile phase: A. acetonitrile: B. water were eluted isocratically with 20% A and 80% B for 10 min at a flow rate of 1 mL/min. Detector used was PDA (Waters 2998), wavelength: 254 nm. Rutin and gallic acid were identified by comparing the UV-Vis spectra and retention times of the peaks and those of the standards. Quantification of the compounds in the extract was carried out by plotting calibration curves of each standard solution with four different concentrations (1000, 500, 250, and 125 μg/mL) versus the areas under the peaks. Precision, linearity, limits of quantification (LOQ) and detection (LOD) were performed to validate the reversed phase HPLC method. Linearity was determined by linear calibration analysis while the calibration curves were used to calculate the correlation coefficient (r 2 ). Inter-assay and intra-assay validation was carried out to determine the precision of the method. LOQ and LOD were calculated from the RSD and slope (S) of the calibration curves using equations, LOQ = 3.3 x (RSD/S) and LOQ = 10 x (RSD/S).

Isolation of polymorphonuclear cells (PMNs), peripheral blood mononuclear cells (PBMCs) and peripheral blood lymphocytes
Ten mL of human whole blood was collected from healthy persons (non-smokers who did not consume medicines or supplements and had fasted overnight) by aseptic vein puncture and placed in heparin-containing tubes. PMNs was isolated from the whole blood by Ficoll gradient separation whereas the isolation of peripheral blood monocytes (PBMCs) and peripheral blood lymphocytes were performed by using lymphoprep as described previously [13,14]. Then, number of monocytes and lymphocytes were adjusted to 5 × 10 5 cells/mL whereas PMNs was adjusted to 1 × 10 6 cells/mL. Approval from the Human Ethical Committee of Universiti Kebangsaan Malaysia (no. FF/2012/Ibrahim/23-May/ 432-May 2012-August 2013) has been obtained for this study.

Cell viability
Cytotoxity of the G. segetum extract and pure compounds on RAW 264.7, PBMC and peripheral blood lymphocytes cells were performed using MTT test [15], while viability test on PMNs was conducted using trypan blue [14].

Chemotaxis
A Boyden chamber was used to perform PMNs migration assay with fMLP (10 −8 M) as a chemoattractant [13]. The working concentrations of the extract and compounds were ranged from 3.125-50 or 6.25-100 μg/mL, respectively. Ibuprofen was used as a positive control, whereas chemoattractant buffer which contain equal amount of HBSS ++ and DMSO was used as negative control. Xylene and haematoxylin fixed and stained the migrated cells and a light microscope was used to measure the cell migration distance.

CD18 integrin expression of leukocytes
The assay was performed using flow cytometry which has been described by Mazzone et al. [16]. Test samples (compounds: 3.125 and 50 μg/mL; extract: 6.25 and 100 μg/mL) and 1 μL of LPS (0.25 μg/mL) were incubated in 100 μL of whole blood in a CO 2 incubator (90 min; 37°C). Then, all the sample tubes were placed in a box containing ice, simultaneously. Immunoglobulin IgG1-FITC (control) or CD18-FITC was mixed into the tubes. The red blood cells were lysed by incubating the tubes in the dark for 20 min after the addition of FACS lysing solution. Then, the mixture was centrifuged (250 g; 5 min at 4°C) and washed with PBS (3×). The CD18 molecules expression was determined by flow cytometry and compared with the control. CD 18 expression was expressed as the percentage of CD18 complex [14,16].

Phagocytosis assay
The effect of G. segetum and its constituent on engulfment activity was perfomed as described in our previous paper [14]. The assay consist of five steps, which include labelling by incubating samples (extract: 6.25 and 100 μg/mL; compounds: 3.125 and 50 μg/mL) with whole blood and E.coli. After incubation, the quenching solution was added to the mixture to quench phagocytosis. Lysing and fixation were done by adding FACS-lysing solution, then centrifuged at 250 g, 2-5°C for 5 min. Then, the leukocytes were washed with PBS and stained with DNA staining solution. The final step was determination of phagocytosis ability of PMNs and monocytes by flow cytometry.

Reactive oxygen species (ROS) production
This assay was conducted according to our previous paper [14]. Samples (compounds: 3.125-50 μg/mL; extract: 6.25-100 μg/mL) were incubated with neutrophils. Opsonized zymosan or PMA was added to induce the cells and luminol (1 × 10 5 M) was used as a probe. Then, the mixture was incubated (37°C; 50 min) in a luminometer. The ROS inhibitory activity of samples was compared with acetylsalicylic acid as positive control and negative control (without sample). The results were expressed as chemiluminescence reading per luminometer unit (RLU) and then calculated as percentage of inhibition.

Nitric oxide (NO) production
The NO production assay was conducted according to a previous paper by Yang et al. [12]. Briefly, RAW 264.7 macrophages cells (1 × 10 6 cells/mL) were seeded in 96well plates for 3 h. Then, cells were incubated wih test samples (compounds: 3.125-50 μg/mL; extract: 6.25-100 μg/mL) or dexamethasone (0.0004 to 4 μg/mL) for another 3 h, then stimulated with LPS (1 μg/mL). After incubation for 24 h at 37°C, 5% CO 2, the production of nitric oxide was determined by measuring the quantity of nitrite in the medium using Griess reagent (0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid and 1% sulfanilamide). One hundred μL of of Griess reagent was added to culture supernatant, then incubated for 10 min in a dark room. A microplate reader was used to measure absorbance at 550 nm and standard solution of sodium nitrite was used to calculate nitrite concentrations.

Cytokine release assay
The enzyme linked immunosorbent assays (ELISA) were employed to determine the effects of the extract and compounds on the release of pro-inflammatory cytokines IL-1β and TNF-α in mononuclear cells, as described by Kumolosasi et al. [17]. The test samples (pure compounds: 3.125 to 50 μg/mL; extract: 6.25 to 100 μg/ mL) or dexamethasone (0.0005 to 5 μg/mL) and LPS (1 μg/mL) were added to PBMCs suspension and incubated for 12 h at 37°C, 5% CO 2. The supernatant was transferred to 96-well plates after centrifugation of the mixtures at 300 x g for 10 min at 4°C The appropriate ELISA-kit was used to assess the cytokine levels in supernatants of human blood cell cultures.

Lymphocytes proliferation
The effect of the extract and pure compounds on lymphocytes proliferation was examined by measuring the incorporation of radioactive thymidine to newly synthesized DNA as written in previous paper [18]. Peripheral blood lymphocytes was incubated with samples (compounds: 3.125-50 μg/mL; extract: 6.25-100 μg/ml) or prednisolone (0.0005, 0.005, 0.05, 0.5, 5 μg/mL) and stimulated with PHA for 3 days in a CO 2 incubator, followed by addition of [ 3 H]Thymidine (0.5 μCi/well). After incubation for 18 h, harvesting was carried out using a cell harvester and a liquid scintillation counter was used to measure [ 3 H]thymidine incorporation as count per minute (CPM).

Statistical analysis
All the data were analysed using Statistical Package for Social Sciences (SPSS) version 15.0. The data were presented as mean ± standard error means (SEM). The IC 50 values was calculated using Graph Pad Prism 6 software. A one-way analysis of variance (ANOVA) for multiple comparisons was employed for data analysis. P < 0.05 was considered to be different significantly.

Quantitative analysis of the methanol extract of G. segetum by HPLC
The chromatogram of the methanol extract of G. segetum showed one major peak of gallic acid and minor peak of rutin, with retention times at 3.078 and 5.161 min, respectively. The peaks were compared to reference standards of gallic acid and rutin (Fig. 2). Quantification analysis showed the amounts of gallic acid and rutin as 191.4 and 24.1 μg/mL, respectively. Correlation coefficients (r 2 ) of   0.9995 and 0.9979 were obtained for gallic acid and rutin, respectively, as the calibration curves were linear over the concentration range of 1000-250 μg/mL. The % RSD values for interday assay precision of peak area and retention time were 3.4 and 0.5% for gallic acid and 6 and 8.6% for rutin, respectively. Whereas the % RSD values for intra-assay precision of peak area and retention time were 3.5 and 0.6% for gallic acid and 6 and 9.1% for rutin, respectively. These values demonstrated the reproducibility of the results. Limit of detection (LOD) and limit of quantification (LOQ) of rutin were found to be 0.44 and 1.34 μg/mL, respectively, while for gallic acid the values were 0.08 and 0.24 μg/mL, respectively.

Phagocytosis activity
The number of neutrophils and monocytes that engulf pathogen were assayed by flow cytometry. Gallic acid showed the lowest inhibitory activity on immunoglobulin and complement opsonized E. coli engulfment by phagocytes. Amongs the samples, 8,8′-(ethene-1,2-diyl)dinaphtalene-1,4,5-triol (4) was the most potent sample in inhibiting phagocytosis ability with percentage of the phagocytizing cells of 44.90 and 47.67% for monocytes and PMNs, respectively. The tubes without sample incubated at 37 o C was used as a positive control while tubes at 0 o C was used negative control (Fig. 3).

Inhibition of reactive oxygen species (ROS) generation
Effects of all the samples on both pathway were investigated using opsonized zymosan and PMA as stimuli. Preliminary evaluation of the samples on the whole blood displayed that all the compounds exhibited strong inhibitory activity upon activation by PMA and zymosan. The samples were further examined for their effects on the respiratory burst of neutrophils. Rutin and 8,8′-(ethene-1,2-diyl)-dinaphtalene-1,4,5-triol (4) revealed similar ROS inhibitory against PMN upon activation by PMA and zymosan with IC 50 values of 0.08 and 0.13 μM for rutin, respectively and 0.13 and 0.05 μM for 8,8′-(ethene-1,2diyl)-dinaphtalene-1,4,5-triol (4), respectively ( Table 3). Table 3 shows the inhibitory activity of standard compounds and isolates from G. segetum on nitric oxide production from RAW 264.7 macrophages cells. All the samples tested revealed significant inhibition with percentage inhibition at highest concentration ranging from 73.86 to 81.57%. As shown in Table 4, 8,8′-(ethene-1,2diyl)-dinaphtalene-1,4,5-triol (4) depicted the strongest NO inhibitory activity with an IC 50 value of 0.15 μM. However, its IC 50 value was higher than that of dexamethasone as positive control (0.025 μM).

Cytokine release
As shown in Table 3, all the compounds tested depicted significant inhibition against proinflammatory cytokines release from peripheral blood mononuclear cells at the highest concentration of 100 μg/mL for gallic acid and 50 μg/mL for others samples. Amongst the compounds, 4,5,4′-trihydroxychalcone (3) demonstrated the highest inhibition on IL-1β release (IC 50 value of 6.69 μM). Meanwhile rutin was the most potent sample against TNF-α release from monocytess with an IC 50 value 16.96 μM. Both compounds might be contributing to the inhibitory activity of G. segetum extract on cytokine release. Table 3 shows the lymphocytes anti-proliferation activity of standard compounds present in G. segetum as well as its isolates. Among the samples, 4,5,4′-trihydroxychalcone (3) was the most potent samples against lymphocytes proliferation (IC 50 value of 1.52 μM) which was comparable to prednisolone (IC 50 value of 0.098 μM) as positive control. Meanwhile gallic acid and rutin, significantly inhibited lymphocytes proliferation after stimulation by a mitogen, PHA (IC 50 values of 0.96 and 2.19 μg/mL, respectively).

Discussion
Phytochemical studies on G. segetum leaves have resulted in the isolation of four compounds. Amongst them, 4,5,4′-trihydroxychalcone (3) is a new naturally occuring compound and this is the first report of its isolation from natural resource, while 8,8′-(ethene-1,2diyl)-dinaphtalene-1,4,5-triol (4) was a new compound. A validated HPLC method was used to quantify the amounts of rutin and gallic acid in G. segetum methanol extract. The cytotoxicity of the plant extract and its constituents was performed using trypan blue and MTT tests. At the highest doses of 50 and 100 μg/mL of G. segetum extract and pure compounds, respectively, the cells showed ≥90% viability.
The ability of G . segetum extracts and its constituents to modulate the immune response was determined by various immunological assays which represent different lineages of immune response. The plant samples were investigated for their effects on chemotaxis, phagocytosis, CD18 expression, and ROS of PMNs, lymphocytes proliferation, cytokine release and NO production of phagocytes. Phagocytic activity consists of several steps, that is, chemotaxis, adherence through the interaction of β 2 integrins with vascular endothelial cells, and engulfment of the pathogen, followed by intracellular destruction. Microbicidal mechanism involves generation of toxic molecules, such as hypochlorous acid (HOCl − ), hydroxyl radicals (OH − ), singlet oxygen and peroxynitrite [20]. Lymphocytes are cells that are most responsible to initiate and perform adaptive immunity. Cytokines also play important role in both innate and acquired immune responses, for example IL-1 and TNF which increase leukocyte/endothelial cell adhesion by increased expression of ligands for integrins [21]. G. segetum and its constituents demonstrated strong inhibition on PMN chemotaxis. The results indicate that 8,8′-(ethene-1,2-diyl)-dinaphtalene-1,4,5-triol (4) was more potent than ibuprofen. Previous study reported the ability of ibuprofen to reduce the interaction between fMLP with its receptor, hence decreasing the migration of PMNs to the site of interaction [22]. The results suggest that G. segetum and its compounds could also decrease the interaction of fMLP with the receptor. The extract and compounds displayed low inhibition of CD18 expression on the surface of phagocytes. According to Luster [23], NO by itself downregulates the expression of integrin on neutrophils, such as CD11a/ CD18 (LFA-1). In this study, all the samples showed strong inhibition on NO production. The less amount of NO might be the reason for low inhibition of the expression of integrin on human phagocytes.
The engulfment activity is initiated by recognition of microbes by phagocytes. Complement surface receptors on neutrophils easily recognize complement-opsonized microbes. Meanwhile, immunoglobulin-coated microbes a b c d e f g are detected by PMNs receptors specific for the Fc-region of immunoglobulin. Interaction of the immunoglobulin and complement with their receptors at the PMN surface initiates phagocytosis of pathogens [24]. The results suggest most of the samples, especially 8,8′-(ethene-1,2-diyl)-dinaphtalene-1,4,5-triol (4) has the ability to block those receptors, hence phagocytosis was inhibited. ROS is produced through independent and dependent receptor pathway. The ROS inhibitory activities of rutin and 8,8′-(ethene-1,2-diyl)-dinaphtalene-1,4,5-triol (4) were higher than aspirin as positive control upon activation by zymosan and PMA (IC 50 values of 0.77 and 10.52 μM, respectively). It was reported that aspirin inhibited luminolamplified chemiluminescence of human neutrophils [25]. Opsonized zymosan stimulated the activity of the enzyme NADPH oxidase by receptor-mediated, whereas PMA activated the enzyme through receptor-independent process [26]. The results suggest all the samples were able to inhibit the ROS production through both pathways. The free phenolic hydroxyl group present in the compounds enhanced the ability to scavenge free-radicals [27].
NO production of macrophages by the enzymatic of inducible nitric oxide synthase (iNOS) has been reduced after exposure to G. segetum extract and its compounds.
Levels of cytokine were increased upon activation of MAPK cascade by LPS which regulates both activator protein-1 (AP-1)-associated gene transcription and NF-ĸB [28]. Another study also reported that corticosteroids act as native inhibitors of IL-l synthesis by mononuclear phagocytes by suppressing biosynthesis of IL-1α and IL-1β at the transcriptional level [29]. Hence, the inhibitory activity of G. segetum extract and its compounds might be due to blockage of biosynthesis pathway at the transcriptional level. IL-1 enhanced CD4+ T-cell proliferation, promoted B-cell growth and differentiation at low locally produced levels [30]. Inhibition on IL-1 release by G. segetum and its compounds consequently inhibited lymphocytes proliferation. Prednisolone was used as a positive control which inhibited lymphocytes proliferation as described previously [31].